the proposed research is designed to obtain a molecular understanding of the mechanisms by which the class of DNA binding proteins called helicases catalytically unwind duplex DNA, in a ATP-dependent reaction, at rates of 500-103 base pairs/sec. this class of proteins is required for replication, recombination and repair processes in E. coli and likely all organisms. In particular, the E. coli rep gene product (Rep) and the E. coli uvrD gene product (helicase II) will be examined using a variety of biochemical and biophysical techniques. Quantitative studies in vitro, of the equilibrium binding of the purified proteins with themselves, single- stranded and duplex DNA, and their nucleotide cofactors will be undertaken as a function of solution variables (temperature, pH, monovalent salt, Mg2+). The effects of these variables on the equilibrium binding constants for the various interactions can be used to obtain thermodynamic information, which is necessary to understand the basis for the stability of these complexes. Kinetic studies of these interactions will also be pursued to understand the mechanisms and pathways of the interactions. In parallel with these studies, the unwinding of various long, duplex DNA substrates will be examined quantitatively to obtain information about the rates and processivities of unwinding. For processively unwinding helicases, the protein must translocate along DNA without dissociating, a process which is of fundamental importance, although we currently understand little about the molecular mechanism. the helicase-catalyzed DNA unwinding reaction will be examined in the absence of DNA synthesis, hence providing a simple system to prove the molecular details of the reaction. These experiments will be used to assess the effects of the f1 gene II protein on the rates of processivity of the REp unwinding reaction, as well as the effects of helix destabilizing proteins. These studies are specifically directed to understand the helicase-catalyzed Dna unwinding reactions; however, the will likely also reveal thermodynamic details that will increase our general knowledge of the basis for stability of protein- DNA complexes. Furthermore, the mechanistic information obtained from the kinetics of these protein-DNA interactions should also aid our understanding of other proteins that must translocate along DNA in order to function (driven thermally by hydrolysis of ATP), e.g., RNA and DNA polymerases. Since DNA replication is fundamental to cell growth in all organisms, an understanding of such a fundamental aspect as the mechanism of enzyme-catalyzed DNA unwinding will undoubtedly have an impact on our understanding of diseases in which replication malfunctions.